1. Field of the Invention
The present invention relates to a method for detecting the suspected presence of a microbial analyte within a sample. More specifically, the present invention relates to an optical detection method using non-specific dyes for detecting the suspected presence of microbial analytes, for example, bacteria, viruses, fungi, rickettsiae and fragments of these microbial analytes among others.
2. Description of the Related Art
There is a requirement for rapid methods of detection and identification of microbial analytes, for example, microorganisms, bacteria, viruses, rickettsiae, fungi and their fragments not only for medical diagnosis, but also for agriculture, food processing, bioprocessing and water purification. Current methods include cell culture, microscopy, immunoassay and nucleic acid probes. Assay times vary from days to minutes. Only culture and polymerase chain reaction (PCR) based tests are very sensitive. Culture and microscopy depend on the isolation of the intact microorganisms from the milieu to be tested, and, for culture, the cells must be viable. Tests based on genetic methods, including polymerase chain reaction, require intact deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The tradeoffs inherent in these various methods are summarized below.
__________________________________________________________________________ Time Sensitivity Specificity Technical Complexity __________________________________________________________________________ Cell culture 1-3 days high, if microorganism moderate simple for bacteria; viable difficult for viruses Microscopy conventional stain hours low moderate moderate selective stain 5 minutes low moderate simple immunofluorescence hours low high moderate immunoassay ELISA (enzyme linked 3-4 hours moderate good moderate immunosorbent assay dip-stick 20-30 minutes moderate good simple latex turbidity 15 minutes moderate good simple gene probe dot blot 1 hour moderate excellent moderate PCR (polymerase chain 2 hours high excellent complex reaction) __________________________________________________________________________
In the immunoassay and immunofluorescence stains previously described, a complex is formed between the antibody, the analyte recognized (from or on the microorganism) and a label or signal generator (i.e. enzyme) that can be measured. The measurement may represent the formation of such a complex, as in sandwich immunoassays, or the lack of formation of such complexes, as in most competitive immunoassays. In a sandwich immunoassay, the label or signal generator is attached to an antibody. There is no direct attachment of the label to the analyte. The binding of the label or signal generator to the analyte is via the antibody.
In a competitive assay, the label or signal generator is bound to an antigen similar to the analyte. As the analyte competes with the labeled antigen for binding to the antibody, the amount of signal changes. In this case also, the label never directly attaches to the analyte.
Other binding molecules besides antibodies have been demonstrated to be useful in sandwich and competitive assays. Such binding molecules include, but are not limited to, lectins, deoxyribonucleic acid (DNA) binding proteins listed by D. J. Kemp, D. B. Smith, S. J. Foote, N. Samaras and M. G. Peterson in Colorimetric detection of specific DNA segments amplified by polymerase chain reactions, PROC. NATL. ACAD. SCI. USA, MEDICAL SCIENCES, Vol. 86, pp. 2423-2427, (April 1989) and by P. K. Sorger, G. Ammerer and D. Shore in chapter eight (8) titled IDENTIFICATION AND PURIFICATION OF SEQUENCE-SPECIFIC DNA-BINDING PROTEINS, in PROTEIN FUNCTION: A PRACTICAL APPROACH, pp. 199-223, (T. E. Creignton, Ed.; (1990), each reference incorporated herein by reference in its entirety and for all purposes. Other binding molecules include receptors and synthetic peptides listed in Random Peptide Libraries: A Source of Specific Protein Binding Molecules by J. J. Devlin et al. published in SCIENCE, Vol. 24, pp. 404-405 (1990), incorporated herein in its entirety by reference and for all purposes.
The use of optical waveguide fibers as a special class of waveguides for immunoassays has been known. For example, U.S. Pat. No. 4,447,546 (Hirschfield, 1984) discloses the use of optical fibers as waveguides which capture and conduct fluorescence radiation emitted by molecules near their surface. U.S. Pat. No. 5,061,857 (Thompson et al., 1991) discloses an optical waveguide-binding sensor having improved sensitivity for use with fluorescence assays.
In the aforementioned patents, the analyte is specifically labeled such that the antibody-analyte complex formed on the optical fiber waveguide is detected due to the fluorescence signal excited and guided toward a fluorimeter using the evanescent wave portion of the optical fiber.
Among the various detection methods, for example, cell culture, microscopy, immunoassay and PCR anasysis, none offer all the advantages of high sensitivity, short assay times (approximately under 5 minutes) and low technical complexity. Furthermore, all of the above patents disclose the use of dyes attached to specific binding molecules (i.e. antibodies or antigens). Assays using dyes bound to specific binding elements may require an incubation time, may be very limited in the amount of the dye that is actually associated with the analyte, and may introduce multi-step procedures into the assay.
In addition to immobilization of antibodies for assay purposes, antibodies on solid supports have been used to bind intact cells and remove them from complex mixtures containing other cell types. Most commonly used forms of this approach are affinity chromatography and panning. In both cases, the sample, containing mixtures of cells, is incubated with the antibody-coated solid support and then the support is gently but thoroughly washed. The bound cells are eluted and subsequently may be subjected to a variety of characterization or experimental procedures. Affinity chromatography and panning techniques are used for isolation purposes rather than for detection of microorganisms or microbial analytes. Such techniques are relatively time consuming, inefficient in terms of cell recovery, and require technical training.